Conversion of red fluorescent protein into a bright blue probe.
We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.
Subach
Oksana M
OM
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Gundorov
Illia S
IS
Yoshimura
Masami
M
Subach
Fedor V
FV
Zhang
Jinghang
J
Grüenwald
David
D
Souslova
Ekaterina A
EA
Chudakov
Dmitriy M
DM
Verkhusha
Vladislav V
VV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003116:Color; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010777:Photochemistry; D016415:Sequence Alignment; D017385:Sequence Homology; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D015335:Molecular Probes; D000097573:Red Fluorescent Protein